Construction Of BMP-6 And VEGF Expression Vector And The Effects To Enhance Bone Formation

Six million fractures occur in the United States with an estimated cost of 20 billion dollars each year. Fractures that do not heal naturally are called non-unions and are surgically treated using various techniques. Large bone defects and non-unions resulting from trauma, tumor resection or other pathologies remains a major clinical challenge. The engineering of bone tissue offers new therapeutic strategies to enhance the regeneration of musculoskeletal tissues. Although considerable research has been performed on bone graft substitutes, the alternatives developed are far from ideal. Recent advances in stem cell biology, gene therapy and osteoconductive matrices have stimulated the development of several composite bone graft substitutes which carry stem cells and growth factors on a suitable matrix. Growth factors capable of promoting bone repair could lead to improved treatment for fracture non-unions, osteoporosis, osteolysis, osteonecrosis and a host of other disorders by allowing for the development of new synthetic graft materials capable of delivering these growth factors safely and efficiently. Bone morphogenetic proteins (BMPs) play a central role in bone regeneration by signaling intracellular pathways that promote osteogenesis. These pathways are responsible for the differentiation of progenitor cells into chondrocytes and osteoblasts, which then modulate new bone formation through endochondral ossification. Several recombinant forms of BMPs, mostly rhBMP-2 and rhBMP-7 (OP-1), have been shown to induce bone formation in vivo, and both rhBMP-2 and rhBMP-7 have been tested in clinical trials. Among the BMPs with osteoinductive potential, previous studies have demonstrated that BMP-2, BMP-6, and BMP-9 (BMP-7 to a lesser extent) are the most potent osteoinductive BMPs. These findings suggest that, in addition to BMP-2 and BMP-7, which are already used in human, BMP-6 and BMP-9 could represent at least equally osteogenic factors for bone regeneration in a clinical setting.Angiogenesis is a fundamental process and a basic condition for tissue regeneration, and vascular endothelial growth factor (VEGF) is the most important regulator of angiogenesis. Previous studies have shown an additive effect of combined delivery of VEGF with BMP-2 or BMP-4 either through cellular delivery or direct delivery of the proteins themselves. Stem cell mediated osteogenesis is more effective as a result of treatment with VEGF plus BMP-4 compared with VEGF plus BMP-2. This suggests that the interaction of VEGF with different BMPs is distinct. Accordingly, this observation may lead to a new strategy for the development of grafts that enhance bone repair as we now know that certain BMPs are more effective than others. Until recently, the effect of combining DNA for VEGF with DNA for BMP-6, has not been evaluated for bone repair. In this study we have chosen BMP-6 over BMP-9 because of evidence in the literature which suggests that VEGF-induced angiogenesis is inhibited by BMP-9. PLAGA microsphere-based scaffold systems fabricated by thermal sintering of individual PLAGA microspheres into 3D structures have been extensively utilized by our group and others. The resulting scaffolds possess a predictable, fully integrated pore network, thereby providing the necessary 3-dimensional microenvironment for cellular infiltration and consequent cell-cell and cell matrix interactions. Our study was based on the hypothesis that VEGF and BMP-6 will act in an additive manner to promote osteoblastic differentiation of D1 cells in vitro and bone formation in vivo. So we constructed expression vector pCMV-hBMP-6 (pB6) and pCMV-hVEGF (pV). We also developed a new strategy for the combined delivery of both VEGF and BMP-6 by using an internal ribosome entry site (IRES) to link the genes that encode BMP6 and VEGF within a single plasmid pCMV-hBMP6-IRES-hVEGF(pVB6) that contains both genes and therefore can express BMP-6 and VEGF simultaneously. We evaluated the effects of BMP-6, VEGF and BMP-6+VEGF on osteoblastic differentiation in vitro with a cloned multipotent mouse osteoprogenitor cell line, D1 cells. Then we plant the constructed recombinant plasmid pB6, pV or pVB6 transfected D1 cells on 3-D Poly(lactic-co-glycolic acid) (PLAGA) scaffolds to construct the tissue engineering biological active bone and to evaluate the BMP-6, VEGF and the synergistic effect of BMP6 and VEGF on ectopic bone formation mediated by D1 cells in vivo.Part 1 Construction of expression vector pB6, pV and pVB6Aim:To construct the expression vector pB6, pV and pVB6 that can express BMP-6, VEGF, BMP-6 and VEGF.Methods:A GFP expressing vector pEGFP-N1 was purchased. The complete human VEGF cDNA segement of 589 bp was PCR amplified from a plasmid containing human VEGF gene. The cDNA segment was then cloned into pCR 2.1-TOPO-TA vector. The clones were confirmed by restriction digestion and complete DNA sequence analysis. The construct pCR2.1-TOPO-hVEGF was digested using restriction enzymes Pvu I and Xho I to cut out a full length VEGF gene and cloned into pShuttle-IRES-hrGFP-1 to generate pShuttle-VEGF. The full length VEGF gene was cut out using Nhe I and Xho I and cloned into expression vector pEGFP-N1 to generate pCMV-hVEGF (pV). Human BMP-6 gene of 1554 bp length was amplified from a plasmid expressing BMP-6 and was cloned as a Nhe I-EcoRV cassette in pShuttle-IRES-hrGFP-1 vector. The full length BMP-6 gene was cut out using Nhe I and Xho I and cloned into pEGFP-N1 to generate pCMV-hBMP-6 (pB6). IRES sequence was PCR amplified from the shuttle vector pShuttle-IRES-hrGFP-1 and subcloned into pShuttle-VEGF using restriction enzymes EcoR V and Pvu I to generate pShuttle-IRES-VEGF. hBMP-6 gene was cloned into pShuttle-IRES-VEGF using the restriction enzyme NheI and EcoRV to generate pShuttle-hBMP-6-IRES-hVEGF. hBMP-6-IRES-hVEGF fragment was restriction digested using Nhe I and Xho I to clone into pEGFP-N1 which resulted in the construct pCMV-hBMP-6-IRES-hVEGF(pVB6). All the constructed plasmids were verified by two enzyme digestion and sequencing.Results:A 589 bp human VEGF,1554 bp human BMP-6 cDNA and IRES sequence was amplified by PCR and the sequencing results showed they were right. The recombinant plasmid pV, pB6 and pVB6 were successfully constructed.Conclusions:The expression vector pB6, pV and pVB6 have been successfully constructed. It provides a sound foundation for further study.Part 2 BMP6 and VEGF expression and the synergistic effect on osteogenic differentiation of D1 cells in vitroAim:To prove the cloned muose osteoprogenitor cell line D1 cell belong to the stem cell. To study BMP-6, VEGF and BMP-6+VEGF expression in transfected D1 cells and BMP-6, VEGF and the synergistic effect of BMP6 and VEGF on osteoblastic differentiation of D1 cells.Methods:First, check the cloned muose osteoprogenitor cell line D1 cell surface markers by flow cytometry to prove it is the stem cell. Six groups for each time point designed for this part:1) BM (DMEM supplemented with 10%FBS),2) OM (DMEM containing 10% FBS with 50μg/ml ascorbic acid,10 mMβ-glycerophosphate, and 10-7 M dexamethasone),3) pN group, pN transfection; 4) pV group, pV transfection; 5) pB6 group, pB6 transfection; and 6) pVB6 group, pVB6 transfection. RT-PCR and ELISA technique was used to detect the expression of target gene. Alkaline phosphatase activity at 2,7 and 14 days was determined using SensoLyte(?) pNPP Alkaline Phosphatase Assay Colorimetric kit. Mineralization of non-transfected and transfected D1 cells after 14 days of growth was determined by von-Kossa staining. Osteocalcin(OCN) and RunX2 gene expression level at 2 days was measured using real-time PCR. The effects of target gene on osteoblastic differentiation of D1 cells were evaluated by the change of ALP activity, cell mineralization and the OCN and RunX2 gene expression level.Results:Flow cytometry results showed that the expression of CD44 and SCA1 are positive, while the expression of CD34 and CD45 are negative. RT-PCR and ELISA results showed that only the cells transfected with one of the three plasmids; pV (expressing VEGF), pB6 (expressing BMP-6) or pVB6 (expressing VEGF and BMP-6) showed considerable mRNA expression or protein expression of respective genes after 2 days of culture. Significant increases in mineralization were observed compared to D1 cells grown without the combination of VEGF and BMP-6. Expression of the RunX2 and OCN genes and the enzymatic activity of alkaline phosphatase increased significantly when VEGF and BMP-6 were expressed together as compared cells expressing BMP-6 alone or VEGF alone.Conclusions:D1 cells belong to the stem cell because of the expression of the cell surface marker CD44 and SCA1. Human BMP-6 and VEGF mRNA and protein expressed in respective plasmid transfected Dl cells. VEGF or BMP-6 can induce osteogenic differentiation of D1 cells in vitro. Futhermore VEGF showed synergistic effect with BMP6 on osteogenic differentiation of D1 cells. Part 3 Construction of delivery of BMP-6 and VEGF tissue engineering bone and the effect on ectopic bone formation mediated by Dl cells in vivoAim:To plant the constructed recombinant plasmid pB6, pV or pVB6 transfected Dl cells on 3-D Poly(lactic-co-glycolic acid) (PLAGA) scaffolds to construct the tissue engineering biological active bone, then to evaluate the BMP-6, VEGF and the synergistic effect of BMP6 and VEGF on ectopic bone formation mediated by Dl cells in vivo.Methods:We constructed the tissue engineering biological active bone by plant the recombinant plasmid transfected D1 cells on 3-D PLAGA scaffolds. We also planted the cells or control plasmid transfected cells or plasmid only on scaffolds as control. We studied the attachment and growth of D1 cells to PLAGA scaffold using Olympus Fluoview laser scanning confocal microscope and scanning electron microscopy (SEM). Blank scaffolds and tissue engineering bone were implanted subcutaneously in Balb/c mice. The mice were randomly divided into 10 groups:(1) PLAGA scaffolds only (P), (2) PLAGA delivering D1 cells only(P+D1), (3) PLAGA delivering pN only (P+pN), (4) PLAGA delivering Dl cells transfecetd with pN (P+D1pN), (5) PLAGA delivering pV only(P+pV), (6) PLAGA delivering D1 cells transfected with pV (P+D1pV), (7) PLAGA delivering pB6 only(P+pB6), (8) PLAGA delivering D1 cells transfected with pB6(P+DlpB6), (9) PLAGA delivering pVB6 only(P+pVB6), (10) PLAGA delivering D1 cells transfected with pVB6 (P+DlpVB6). Implants (n=4) were retrieved at 2,3, and 4 weeks after implantation. Bone and blood vessel formation of all implants were determined qualitatively and quantitatively by methods including histology, immmunostaining, X-ray and Micro-CT. Results:Olympus Fluoview laser scanning confocal microscope and SEM images showed that D1 cells attach and grow very vell on the PLAGA scaffold. X-ray and Micro-CT analysis of the retrieved implants over time revealed the synergistic effect of combining VEGF with BMP-6, as greater ectopic bone formation was observed in the VEGF plus BMP-6 group compared to the VEGF alone or BMP-6 alone at 2 and 3 weeks, but at 4 weeks, there is no significant difference between P+D1pVB6 group and P+D1pB6 group. Micro-CT showed that mineralization was present mainly at the peripheral region at 3 weeks, whereas at 4 weeks, the mineralization was evenly distributed throughout the scaffold volume. The HE staining and von-Kossa staining were in accordance of the Micro-CT data. We observed overall more cellularity and more mineral deposition in the PLAGA implants carrying D1 cells expressing VEGF and BMP-6 at 3 weeks time point. Blood vessel staining and HE quantification revealed an increase in the number of blood vessels within the implants that were treated with D1 cells that expressed VEGF.Conclusions:The 3D-PLAGA scaffold supports the attachment and growth of both transfected and non-transfected D1 cells. New tissue engineering biological active bone was constructed by plant the recombinant plasmid transfected D1 cells on 3-D PLAGA scaffolds. X-ray and Micro-CT analysis showed that VEGF or BMP-6 can induce osteogenesis mediated by D1 cells in vivo. Furthermore, VEGF demonstrated synergistic effect with BMP6 on ectopic bone formation mediated by D1 cells in vivo. Histology results showed that VEGF can promote angiogenesis, but can promote mineral deposition and bone formation combined with BMP-6.